1. Field of the Invention
This invention relates generally to methods and a system for the separation and resolution of molecular components, including synthetic, organic or biologic particles, from a mixture of such components. More particularly, the invention pertains to a multilayer molecular filtration system comprised of discontinuous and nonsequential, gelous, semifluid or fluid, chromatographic or electrophoretic mediae placed on or inside or around different and presently known and used mechanical vectors, facilitating a movement of polar particles with various molecular characteristics and related methods thereof.
2. Description of Related Art
The use of sieving gels for the separation of organical and/or biological particles is well-known and used in the electrophoresis art from mid-fifties of this century. However, the presently known methods, based on sieving effects demonstrated throughout the years, have serious disadvantages. The most apparent is the inability to separate, satisfactorily, the high and very high molecular particles, e.g., molecular weights greater than 650,000 daltons.
The sieving gels which are most often used for analytical and scientific purposes include the following: cellulose acetate, starch gels, agar-gels, agarose-gels, gels of agarose derivatives, polyacrylamide gels, gradient polyacrylamide gels of different gradients (from 2% to 27% of PAA and 4% to 30% of PAA), pore gradient polyacrylamide gels, mixture of, or combinations of polyacrylamide gels and different derivatives of agarose, as well as any of the above gels with addition of protein-complexes-forming variants (SDS), urea, or emulators (TRITONS), or glass adhesives, etc.
All the above-mentioned gels and combinations thereof are known to be used only in continuous forms (e.g., nongradient mediae), or continuous gradients (e.g., gradient mediae). For example, the most often employed gelous mediae are the agarose-gels. Although gels of different concentrations of purified agarose are utilized for specific purposes (e.g., 1%, 11/2%, 2%, 21/2%, etc.), all are known as "continuous" gels. In comparison, the "gradient" gels comprise gels (usually polyacrylamidic) of increasing concentrations of acrylamide monomer, as shown by the formula where T equals the percent of monomers concentration: ##EQU1##
These gels are known to be used in different ranges of their gradients (for instance from 3-7%, or 2-5%, or 2-16%, or 2-27%), but these gradients are known to be always sequentially assorted (e.g., in 3-4-5-6-7%, or 2-3-4-5%, or in 2-3-4-5-6-12-13-14-15-16% fashion, etc.).
It is noted that the literature discloses only few devices of similar arrangement of two electrophoretic gels from which two gels designated (A,B) were called "discontinuous", and one designated (C) was described as a gel with a "stepped gradient ". However, none of the above arrangements suggest a variable multilayer system of distinct electrophoretic gels, forming specific molecular sieves characterized by retention gradients for elected groups of polar biomolecules.
The above designated devices (A, B and C) only comprise two (2) layers of gels wherein the first layer serves primarily as a mechanical, and not as an analytical means (e.g., delivery of the biological sample to an analytical gel (A), or to clean the analyzed biological sample from interfering components (B), or to unspecified improvement of desired analytical procedures (C)). In addition, certain ones, called "technical" gels are composed of components which are substantially smaller, than are the main, analytical gels (A, B, C).
U.S. Pat. No. 4,306,956 to De Castro describes the first of the two-layer systems designated (A). This patent is directed to the use of thymol blue, phenol red, o-cresol red, orange G, m-cresol purple and mixtures, as a tracking dye in an SDS-PAGE electrophoresis process. The patent describes system designated (A) as follows--"Often in discontinuous sodium-dodecyl sulfate polyacrylamide electrophoretic gels (U.S. Pat. No. 3,384,564), two separately polymerized layers of polyacrylamide, designated as `a stacking gel` and `a separating gel`, are prepared."(Col. 1, lines 40-42). The large pore size of "the stacking gels" allows the biological sample to concentrate into narrow starting zone, necessary for good resolution in the following gel, with no change in characteristics, until it encounters a discontinuity in entering "the separating gel", either in the nature of the supporting medium, i.e., pore size, or in the buffer, e.g., pH. This change, based on entering into second gel, produces the separation of the different macromolecular species into discrete bands, but it does not relate to the present invention.
The second of the two-layer systems (B) was also called "discontinuous" by S. Moulin, J. C. Fruchart, P. Dewailly and G. Sezille--Electrophorese des lipoproteines serigues sur plaque de gel d'acrylamide-agarose, en gradient discontinu d'acrylamide. Clinca Chimica Acta, 91 (1979): 159-163. The authors used two different concentrations (2% and 3%) of acrylamide and a constant concentration of agarose (0.7%). The biological samples are placed in the first gel (2%) in order to retain macromolecular components (e.g., chylomicrons), which hinder a good electrophoretic separation of other lower molecular components of diagnostic interest. However, the entire analytical separation takes place on a homogeneous second gel (3%), without any further interference of the first layer which clearly serves only as a technical preserver of the unwanted material and therefore does not relate to the present invention.
U.S. Pat. No. 4,189,370 to Boschetti describes the third of these two-layer systems (designated (C)) which was referred to as a "stepped gradient". The patent is directed to a process for preparing gels stratified upon a film, comprising two steps: first, disposing upon said film gel, being formed from a first monomer concentration and disposing upon said first film a second gel, so as to form "a discontinuous gradient" between said first gel and second gel; these layers may be spaced from polyester film and the second layer may also be disposed on said first layer. The author failed to demonstrate how the separation of biomacromolecules is enhanced by said invented separation line (between layers). However, the so claimed, but not proven "separation" may be often artificial, or otherwise incorrect, or may be related to wrongly defined fractions, etc.
The ideas of Boschetti (1980) and de Castro (1981) are basically identical, although their inventions differ in concentrations of recommended gels for electrophoresis of serum lipoproteins and/or proteins--de Castro's so called "stacking gel" has lower T% than his "separating gradient gel" offered in a wide variety of gradually increasing compositions from 0.5% to 30% T; preferred range is from about 3-27% T. similarly, Boschetti's first gel has lower concentrations of said bi-functional allylic or acrylic compound e.g., about 7.5%, while the nongradient second gel has higher concentrations, e.g., about 10-17%, although concentrations of the monomers in the said gels differ not significantly, e.g., 2.5% and 2.0%, respectively.
However, none of the above-described methodical modifications of separating gels make possible to retain and so separate the analyzed biomolecules according to their specific retention gradients into their standard classes and subclasses. This is described in detail herein, under EXAMPLE 1 and graphically presented in FIG. II wherein a pattern obtained with de Castro's method (classified as GRAD) can be compared with the method of this inventor (classified as DISC).
U.S. Pat. No. 3,384,564 to L. Ornstein is directed to "a simultaneous separation and concentration of charged particles which may be either separately recovered or alternatively the resulting continuous thin zones of charged particles used thereafter as the starting zones with appropriate pH and/or pore size change for high resolution and further separation of the components in brief runs." (Column 1, lines 10-19) The patent states that "with a synthetic polymer like polyacrylamide, because the average pore size of a gel depends on the concentration of polymer (e.g., a 30% gel produces about a 20 .ANG. pore and 71/2% about 50), the pore size can be adjusted to the range of dimensions of the molecules t o be separated" and gives the dimensions in .ANG. and the free mobilities of a few plasma proteins at pH 8.6 (mobility unit=10.sup.-5 cm.sup.2 /volt-sec.). (Column 1, line 70 --column 7, line 5)
L. Ornstein stated his basic working idea as follows: "It would appear that a 71/2% aqueous polyacrylamide should exhibit extreme frictional resistance to the migration of fibrinogen, .beta..sub.1 lipoprotein (and perhaps the .alpha., macroglobulin and /gamma/globulin), and that the other proteins should be able to pass through, though with substantially more difficulty than in an aqueous system. Thus differences in molecular size as well as charge can be utilized in electrophoretic separation of various components in a gel or solution of linear polymer."
L. Ornstein's claims of an electrophoresis, using three layers, although in fact only two real layers are described by him--a large pore specimen layer (it is not presently considered as a separate layer), large pore gel spacer layer (later called "stacking gel") and a small pore gel layer for further separation (=on 7.5% PAAG) of analyzed particles in a vertical cylindrical container, equipped with buffer reservoirs and electrodes, fraction elutor and sample collector. Thus, Ornstein does not relate to the present invention.
Commercially, the combination of a "stacking and separating gel" has been utilized according to de Castro by Miles Laboratories, Elkhart, Ind., under the trademark "LipoPhor-Gels". They were primarily used for HDL-cholesterol assaying by D. Roche, V. Atger, N. T. Le Quang, A. Girard, and O. G. Edindjian in the work "Polyacrylamide Gel Electrophoresis in Quantification of High-Density Lipoprotein Cholesterol", Clinical Chemistry, 31/11 (1985), 1893-1985. The authors found good correlations between total HDL-cholesterol analyses and corresponding ultracentrifugal values, but never attempted to achieve the separation of HD-lipoproteins according to their molecular sizes. Their work does not interfere with this invention.
More sophisticatedly were removed from the biologic mixture of serum lipoproteins the VLDL (and chylomicrons) by the accumulation at the 5 mm thick 2%, 5% PAAG boundary, while LDL just entered the 5% PAAG. The separation between the above lipoprotein groups of VLDL and LDL thus obtained "was variable, and not sufficiently clear for analytical purposes." "The effect of the 2% and 5% gel phases was therefore to retard the LDL and VLDL over some distance of the gel system without compressing these into a thin concentrated band at a gel boundary", according to the authors J. B. Ubbing, W. J. Serfontein and L. S. devilliers in an article "The Direct Quantification in Whole Serum of HDL Subfractions", Clinica Chimica Acta, 125 (1982), 165-175.
These authors achieved the above-described "stacking effect" of HDL for some kind of separation of the HDL into HDL.sub.2 and HDL.sub.3 subfractions, roughly corresponding to the ultra-centrifugally isolated HDL classes at the density 1.125 g/ml (HDL.sub.2) and 1.21 g/ml (HDL.sub.3), using an additional 3 mm thick polyacrylamide gel (PAAG), dimensions 160.times.120 mm, consisting of a 13% gel of classical composition at pH=8.9.